Extensive analysis of the FMR-1 gene and the fragile X mutation site in human Xq27.3 is proposed. This gene is likely to be involved in the phenotypic features of fragile X syndrome, the most common form of inherited mental retardation in humans, with a frequency in boys of approximately 1/1200. Analysis of the spectrum of mutations in fragile X families will provide insight into the very unusual mutational and genetic mechanisms in this peculiar region of the X chromosome. Analysis of the FMR-1 gene and its RNA and protein products will allow determination of its role in normal development. The consequences of defects in this gene and their role in generation of mental retardation will be studied. Understanding the basis of the fragile X phenotype will provide insight into potential therapies, and into the nature of other mental deficiencies. The specific aims of the study are: 1) Characterization of the fragile X mutation site in genomic DNA from patients and other family members; 2) Characterization of the FMR-1 gene located at the fragile X site by definition of its structure, isolation of cDNA representing its mRNA, development of antisera specific to its protein product, and use of these tools to study its expression and presence in tissues from normal and fragile X humans as well as mice at various stages of development; 3) Isolation of the murine equivalent of the FMR-1 gene, characterization of its expression and of the region of the mouse X chromosome for the presence of the peculiar CGG repeat sequence found in humans; 4) Production of a mouse model of fragile X syndrome by gene targeting of the FMR-1 gene in embryonic stem cells and creation of chimeric animals derived from the mutant ES cells; once established, the mouse models will be extensively studied for phenotypic features similar to human fragile X syndrome; 5) Study of the fragile X region for additional genes, and characterization of these for their role (if any) in the syndrome; 6) Identification of other sites in the human genome with long stretches of the CGG repeat sequence, and study of their locations relative to other fragile sites; 7) Analysis of the FMR-1 gene for its role in other, non-fragile X related, mental deficits by mutation detection methods. Characterization of the fragile X mutation and its associated gene will lead to improved diagnosis and provide the capacity to design therapies for this common form of inherited mental retardation. Analysis of the gene should also provide insight into brain function and mechanisms of retardation.